Use of surface plasmon resonance for real-time measurements of the global conformational transition in human phenylalanine hydroxylase in response to substrate binding and catalytic activation

In the present study the optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the reversible binding of the pterin cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH,) and L-phenylalanine (L-Phe) to human phenylalanine hydroxylase (hPAH), When BH4 (241 Dal was injected over the sensor chip with immobilized tetrameric wt-hPAH a positive Delta RU response was observed with a square-wave type of sensorgram and a saturable response (about 25 RU/(pmol subunit/mm(2)) with a [S](0.5) value of 5.6 +/- 0.8 muM for the pterin cofactor. The rapid on-and-off rates were, however, not possible to determine. By contrast, when L-Phe (165 De) was injected a time-dependent increase in RU (up to about 3 min) and a much higher saturable Delta RU response (about 75 RU/ (pmol subunit/mm(2)) at 2 mM L-Phe) than expected (i.e,, <5 RU/(pmol subunit/mm(2))) from the low molecular mass of L-Phe were observed in the sensorgram. The half-time for the on-and-off rates were 6 +/- 2 and 9 +/- 1 s, respectively, at 2 mM L-Phe, The steady-state (apparent equilibrium) response revealed a hyperbolic concentration dependence with a [S](0.5) value of 98 +/- 7 muM. The [S](0.5) values of both pterin cofactor and L-Phe were lower than those determined by steady-state enzyme kinetic analysis. Evidence is presented that the Delta RU response to L-Phe is accounted for by the global conformational transition which occurs in the enzyme upon L-Phe binding, i.e., by the slow reversible transition from a low activity state (T-state) to a high activity state (R-state) characteristic of this hysteretic enzyme. (C) 2001 Academic Press.